F O O D B I OT E C H N O L O G Y

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1 I L S I E U R O P E C O N C I S E M O N O G R A P H S E R I E S F O O D B I OT E C H N O L O G Y AN INTRODUCTION

2 FOOD BIOTECHNOLOGY AN INTRODUCTION by Dean Madden ILSI Europe

3 ILSI EUROPE CONCISE MONOGRAPHS This booklet is one in a series of ILSI Europe concise monographs. The concise monographs are written for those with a general background in the life sciences. However, the style and content of these monographs will also appeal to a wider audience who seek up-to-date and authoritative reviews on a variety of important topics relating to nutrition, health and food safety. Concise monographs present an overview of a particular scientific topic and are usually based on proceedings of scientific meetings. The text of each concise monograph is peer reviewed by academic scientists of high standing. The concise monographs make important results and conclusions available to a wider audience. The following titles are available in the series: Starches and Sugars: A Comparison of Their Metabolism in Man A Simple Guide to Understanding and Applying the Hazard Analysis Critical Control Point Concept Food Allergy and Other Adverse Reactions to Food Dietary Fibre Oxidants, Antioxidants, and Disease Prevention Caries Preventive Strategies Sweetness the Biological, Behavioural and Social Aspects. Concise monographs in preparation include: Dietary Fat Some Aspects of Nutrition and Health and Product Development; The Nutritional and Health Aspects of Sugars; Nutritional Epidemiology: Limits and Possibilities. INTERNATIONAL LIFE SCIENCES INSTITUTE AND ILSI EUROPE The International Life Sciences Institute (ILSI) is a worldwide, non-profit foundation headquartered in Washington, D.C., USA, with branches in Argentina, Australia, Brazil, Europe, Japan, Mexico, North America, Southeast Asia, and Thailand, with a focal point in China. ILSI is affiliated with the World Health Organization as a non-governmental organization (NGO) and has specialised consultative status with the Food and Agriculture Organization of the United Nations. ILSI Europe was established in 1986 to provide a neutral forum through which members of industry and experts from academic, medical and public institutions can address topics related to health, nutrition and food safety throughout Europe in order to advance the understanding and resolution of scientific issues in these areas. ILSI Europe is active in the fields of nutrition and food safety. It sponsors re s e a rc h, conferences, workshops and publications. For more information about its programmes and activities, please contact: ILSI Europe Avenue E. Mounier 83, Box 6 B-1200 BRUSSELS Belgium Telephone (+32) Telefax (+32)

4 The use of trade names and commercial sources in this document is for purposes of identification only, and does not imply endorsement by the International Life Sciences Institute (ILSI). In addition, the views expressed herein are those of the individual authors and/or their organisations, and do not necessarily reflect those of ILSI. Copyright 1995 by the International Life Sciences Institute All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright holder. ILSI Press 1126 Sixteenth Street, N.W. Washington, D.C USA Telephone: (+1) Telefax: (+1) ILSI Europe Avenue E. Mounier 83, Box 6 B-1200 Brussels Belgium Telephone: (+32) Telefax: (+32) Printed in Belgium ISBN

5 FOREWORD The professor to his cook: You are a little opinionated and I have some trouble making you understand that the phenomena which take place in your laboratory [kitchen] are nothing other than the eternal laws of nature, and that certain things which you do without thinking, and only because you have seen others do them, derive nonetheless from the highest scientific principles. Jean Anthelme Brillat-Savarin The Physiology of Taste, 1825 The sentiments expressed by Brillat-Savarin over a century ago are even more relevant today as the technology associated with food production, processing and preparation becomes increasingly complex. Traditional biotechnology brewing, baking and other fermentation processes has been associated with food for thousands of years. Now modern biotechnology, and genetic modification in particular, is beginning to make an impact. Given food's deep cultural importance, it is hardly surprising that this has created anxieties. This monograph, which aims to address those concerns and to describe the opportunities presented by modern biotechnology, is in five sections. In the first two parts the technology of genetic modification is explained. Numerous examples of its use in food production are described in the third section. Several case studies then illustrate how enzymes are used in food processing, showing how many modern practices originated from ancient traditions. Finally, some of the social, economic and safety implications of genetic modification are examined. Author: Dean Madden Scientific Editors: David A. Jonas, Axel Kahn, Michael Teuber Series Editor: Nicholas J. Jardine

6 CONTENTS What is biotechnology?... 1 Methods used in biotechnology... 2 Genetics and genetic modification... 2 Other biotechnological methods Food production Microbial production of food, food additives and processing aids Plant biotechnology Animal biotechnology Examples of the use of enzymes in food processing Cheese manufacture Fruit juice production Sweetener production Regulatory, safety and socioeconomic considerations Food safety Social, economic and other effects The need for public involvement Bibliography... 37

7 Food Biotechnology 1 WHAT IS BIOTECHNOLOGY? The term "biotechnology" was first coined by a Hungarian, Karl Ereky, towards the end of World War I. Ereky used the word to refer to intensive agricultural methods. Since that time biotechnology has been variously defined, but it has nearly always been associated with food production and processing. In particular biotechnology has usually encompassed the traditional manufacture of bread, wine, cheese and other fermented foods. On these grounds, biotechnology can trace its roots back several thousand years to the ancient Sumerians, who brewed beer with naturally occurring yeasts. Ancient fermentations were not always successful. The microbes that fell into the wine maker's vat could yield the finest vintage or transform the entire product to vinegar. In the 1800s Louis Pasteur laid the foundations of microbiology and identified micro o rganisms bacteria, fungi, algae and protozoa as the cause of both desirable and undesirable changes in food. The application of Pasteur's research led to safer, more reliable food processing and preservation and helped ensure the consistent high quality of, for example, fine wines and cheeses. Pasteur asserted that fermentation processes were inextricably linked to the activities of living microbes. Towards the end of the last century it was discovered that cell-free extracts from yeast could also bring about chemical changes without the intervention of the microbes from which they were derived. The active components of such extracts were named enzymes (enzyme means "in yeast"). Enzymes are proteins, made by all living things, that catalyse specific chemical reactions. Without realizing it, the makers of cheese had always used a mixture of natural enzymes rennet to transform milk into solid curds and liquid whey. During the 1940s, large-scale fermentation equipment was developed which led to the efficient industrial production by microorganisms of pure enzymes and additives and other valuable compounds (such as vitamins) for use in food. Just as different breweries have their own carefully maintained proprietary strains of yeast, enzyme manufacturers culture specially selected strains of their chosen micro o rganisms. Over many years gre a t improvements have been made to the efficiency of p roduction and the safety, quality and range of m i c robial products available. However, much still depends on chance occurrence followed by systematic isolation of organisms with desirable characteristics. With the advent during the 1970s of the ability to make precise changes to genetic material, biotechnology was transformed. The performance of organisms can now be "fine tuned", and biotechnology has now almost became a synonym for "genetic modification". In 1980, an influential British report (the "Spinks Report") attempted to encapsulate nearly half a century of European and United States thought, defining biotechnology as "the application of biological organisms, systems or processes to the manufacturing and service industries". This broad definition suits our purposes, as it includes the production of food by living organisms, its subsequent processing with the assistance of microbes or enzymes and the assurance of food quality and safety using the tools of molecular biology.

8 2 Concise Monograph Series METHODS USED IN BIOTECHNOLOGY Genetics and genetic modification Chromosomes, genes and DNA Genes, passed from one generation to the next, determine all inherited characteristics. Genes are made from DNA (deoxyribonucleic acid), most of which is packaged, in fungal (including yeast), plant and animal cells, into chromosomes within the nucleus of the cell (Figure 1). Some genes are also found outside the nucleus: in the mitochondria (which release energy for cellular activities) and within the chloroplasts (sites of photosynthesis) of plant cells. In bacteria, most genes occur on a single circular chromosome, although small rings of DNA called plasmids may also be present. The double helix of DNA can be likened to a twisted rope ladder. The two intertwined helices are chains made from sugar and phosphate molecules linked together alternately. Attached to each sugar molecule is a "base". There are four different bases: adenine (A), thymine (T), cytosine (C) and guanine (G). Weak bonds between the bases join the two strands of the double helix together like the rungs of a ladder. A always pairs with T, and C always pairs with G. This "base pairing" mechanism ensures identical replication of DNA strands during cell division (Figure 2). A particular gene (a stretch of DNA with a particular sequence) determines the structure of all or part of a specific protein (Figure 3). The sequences of bases in the DNA specify the amino acid residues that are needed to make proteins. Three bases in a row specify each amino acid, and the sequence that specifies each the genetic code is the same in all living organisms (see Figure 2). Also encoded within the DNAare instructions to regulate protein production. Although all cells of an organism will contain the same DNA, only certain proteins will be made at any one time or in any particular type of cell; that is, only certain genes will be expressed. DNA has an identical structure in all living things, and because the genetic code is universal, the possibility is raised that genes can be transferred between completely different species. The process of transferring, removing or altering genetic information by the modification of DNA is commonly called genetic modification (or genetic engineering). Why alter nature? In nature, proteins are often made in minute quantities and are therefore difficult or impossible to extract and purify. These proteins include enzymes and a variety of pharmacologically active compounds such as insulin for diabetics, interferons for cancer therapy and vaccines to help prevent diseases. Often large quantities of such valuable proteins are needed. Biotechnologists can achieve this by transferring the relevant genes into microbes that can easily be cultivated in large numbers. The same technology, applied to the production of food, could bring significant benefits. I m p roved varieties for agriculture. Animal and plant breeders have for centuries selected livestock and plants with desirable characteristics. Breeding from chosen stock is a very slow process that can be set back by the chance recombination of genes in the offspring. A breeder may select a preferred trait, only to find that it is accompanied by an equally undesirable one, which then has to be painstakingly bred out. Traditional methods of selecting the best plants or animals from which to breed have been greatly aided by modern genetic techniques. Furthermore, it is now

9 Food Biotechnology 3 FIGURE 1. The essential structure of bacterial, animal and plant cells Bacterial cell chromosome plasmids, small rings of DNA FIGURE 2. The structure of DNA, the hereditary material. Three bases in a row code for amino acids that a re the "building blocks" of pro t e i n s. DNA structure 0,5 µm ribosomes sites of protein synthesis cell wall cytoplasm cell membrane nucleotide Animal cell nucleus cell membrane chromosomes (made from DNA + protein) 15 µm ribosomes cytoplasm mitochondria, sites of energy release (have their own DNA) First position Second position Third position hydrogen bonds base sugar phosphate Plant cell chloroplast, site of photosynthesis (has its own DNA) cell membrane m i t o ch o n d r i o n The genetic code cytoplasm vacuole 30 µm cell wall ribosomes nucleus, containing chromosomes

10 4 Concise Monograph Series FIGURE 3. Protein synthesis DNA messenger ribonucleic acid (mrna) amino acids protein amino acid chain gene bases mrna ribosome amino acid chains DNA determines hereditary characteristics by directing the formation of particular proteins. A stretch of DNA carrying the instructions required to make a particular protein is called a gene. DNA operates through an intermediary, RNA. The sequence of bases on the DNA molecule acts as a template for the production of messenger RNA (mrna). The mrna binds to the ribosomes, where it directs the production of the protein it encodes. Three mrna bases in a row specify each amino acid to be added to the protein. The amino acid chain(s) that proteins are formed from fold into precise threedimensional structures, contributing to the protein s properties and function. possible to make very precise changes to the genetic material. This can help improve the resistance to disease and environmental stress amongst crop plants and farm animals. It can also help boost agricultural productivity and enhance the nutritional status, storage properties and ease of processing of food products. Food additives and processing aids. Enzymes are specialized proteins that are essential for life. They catalyse all biological processes and thus contro l metabolism in living organisms. Once extracted from living organisms, these proteins allow certain processes in food production to be conducted. For thousands of years enzymes such as rennet from animals and papain from plants have been used to enhance the flavour, texture and appearance of food. Because of the diversity of microorganisms, it has been possible to find a wide range of microbial enzymes that are active in the conditions encountered in food processing. With genetic modification a greater range of pure and highly specific enzymes can be produced more eff i c i e n t l y. These enzymes can be used to make desirable changes to food both rapidly and at relatively low temperatures, with a subsequent reduction in fuel requirements and in the e n v i ronmental impact of food processing. To the consumer, the direct benefits include better flavour, texture and shelf life of food, often with a reduction in the need for processing and additives.

11 Food Biotechnology 5 FIGURE 4. Restriction enzymes "recognise" and cut DNA molecules at precise locations. The names of these enzymes are derived from those of the organisms that produce them. The sequences of bases that are recognised by three enzymes (BamHI, HindIII and EcoRI) are shown here. BamHI HindIII EcoRI How genetic modification is done Cutting and pasting DNA. Special enzymes, obtained from bacteria, are an essential tool of the molecular biologist. In nature, these enzymes help bacteria fend off viral attack by precisely dissecting the foreign DNA of invading viruses. In this way, the proliferation of the viruses is restricted. Restriction enzymes (as they are known) recognize and cut DNA molecules at specific locations (Figure 4). Many hundreds of re s t r i c t i o n enzymes have been isolated from different microbes and are available commerc i a l l y. With re s t r i c t i o n enzymes almost any section of DNA, and consequently any single gene, can be excised at will. The end of one DNA molecule will readily link to that of another that has been cut with the same enzyme. To join two DNA molecules permanently it is necessary to form chemical bonds along the DNA's sugar-phosphate backbone. An enzyme called DNA ligase can do this job. The function of these "cut and paste" enzymes in assembling novel DNA molecules is obvious, but the genetic engineer's tool kit would be incomplete without one or two other enzymes. To understand their role it is necessary to appreciate how proteins are made. A genetic intermediary. The genetic information encoded in DNA lies within the nucleus of the cell. However, proteins are not made in the nucleus, but elsewhere, at special structures called ribosomes. Before a particular p rotein can be made, a copy of the appro p r i a t e instructions must first be transcribed from the DNAand then ferried to the ribosomes. The copied instructions are made from mrna (messenger ribonucleic acid). This mrna is virtually a mirror image of the sequence of bases on one DNA strand, according to the basepairing rules. Upon arrival at the ribosomes the base sequence within the mrna directs the construction of proteins from amino acids. A sequence of three adjacent bases in the mrna molecule is needed to determine each amino acid in the protein (see Figure 3). Cells that are producing a particular protein will have many identical copies of that protein's mrna inside them. It is often easier to search for genes among the small mrna molecules rather than along the entire length of the cell's DNA. Once a desired length of mrna has been isolated, two additional enzymes are needed.

12 6 Concise Monograph Series D N A f rom RNA. The enzyme reverse transcriptase assembles a single strand of complementary DNA alongside a corresponding piece of mrna. A second enzyme (DNA polymerase) can then be used to construct a double-stranded helix using the first DNA strand as a template. DNA made in this way is called copy or complementary DNA(cDNA). A copy of a gene from a donor cell is used in genetic modification. Gene synthesis. By the judicious use of restriction and other enzymes, molecular biologists are able to assemble DNA molecules which contain one or more genes of interest. Where a particular piece of DNA is difficult to isolate, it is sometimes possible to make it artificially using a DNA synthesizer. Under computer control, these devices string together the biochemical precursors needed to make short stretches of DNA. Of course, to programme the synthesizer it is necessary to know the sequence of bases present in the desired gene; this too can be determined automatically using a DNA sequencer. It is also possible to copy specific genes using the polymerase chain reaction (PCR). The PCR has been likened to a "genetic photocopier". From a very small amount of DNA millions of copies of a specific section of DNAcan be made quickly. The PCR lies behind many of the spectacular successes of forensic genetic fingerprinting, where criminals have been identified from the DNA in just a few drops of blood or even a couple of cells on a cigarette butt. P l a s m i d s. Once a suitable DNA molecule has been constructed, it must be moved into a cell in which it can be expressed and duplicated so that it passes from one cell division to the next. For microorganisms, one of the most successful methods involves the use of plasmids as a vehicle for transferring genes. Plasmids are rings of DNA that are found in some cells. They carry a limited set of genes and normally constitute only a few percent of a cell's total DNA. During the course of evolution, plasmids carrying genes that help their microbial hosts survive have been selected by nature. Some plasmids confer on their hosts the ability to degrade substances in the environment such as nutrients and antibiotics. Many traditional foods, such as yoghurt, cheese and other fermented dairy products, contain large numbers of living microbes that naturally harbour plasmids. Like the DNA of chromosomes, that of plasmids can be cut with restriction enzymes and additional DNA pasted into it. The result is a ring of "recombinant" DNA that can be put into a bacterium. Specialized plasmids can be used to ferry genes from bacteria into yeast cells or even plants (Figure 5). A limitation of plasmids is that they cannot accommodate DNAfragments longer than base pairs. However, some harmless, specially tailore d v i ruses can package larger DNA molecules. Such viruses have been used to transfer genes into microbes, plants and animals and even to treat human disease. Genetically modified plants. A vector system that is used for a wide variety of plants is the plant tumour-inducing plasmid (Ti-plasmid) found in the soil bacterium A g robacterium tumefaciens. Through its plasmid, Agrobacterium has the ability to naturally engineer plant cells so that they grow tumours that pro d u c e compounds which the bacteria need to sustain themselves. Molecular biologists use disarmed (nontumour-inducing) versions of this plasmid to introduce foreign genes of their choice into plants. Because every cell carries a complete copy of all the plant's genes in its chromosomes, it is possible to regrow an entire plant from a single modified cell (see Cell culture, below). Specially modified Ti-plasmids have now been produced which help transfer fairly large genes into plants. Unfortunately, monocotyledons (including the important cereal crops) are resistant to Agrobacterium.

13 Food Biotechnology 7 FIGURE 5. Pure chymosin for cheese making can now be obtained from genetically modified yeast DNA Copy of chymosin gene Sample of calf cells Chymosin from modified yeast cells Chymosin gene inserted into plasmid Calf Plasmid put into yeast cells A g ro b a c t e r i u m has proved especially useful when working with trees, which, because they are slow g rowing and large, are difficult to improve by conventional breeding. Apricot, plum, apple and walnut trees have all been genetically modified with Agrobacterium (Figure 6). Gene ballistics. A procedure called ballistic bombardment has achieved success with several crops, including rice, wheat and soya. With this method, the DNA to be introduced into the plant cells is first stuck onto minute tungsten or gold particles. The DNA-coated particles are fired at high velocity into soft plant tissue, usually callus (see Cell culture, below). This intro d u c e s functional DNA into the plant cells. Electroporation. DNA can also be introduced into the thin-walled tubes which develop from pollen grains by subjecting them to microsecond pulses of a stro n g electric field. This technique, called electro p o r a t i o n, causes pores to appear momentarily in the pollen tubes through which DNA from a surrounding solution can enter. Seeds that develop from ovules fertilized with such pollen carry the introduced genes. Electroporation also works with plant cells from which the cell wall has been removed by enzyme treatment. From these naked plant cells, whole plants can be regenerated by cell culture (see Cell culture, below). Electroporation has also been used to transfer DNA molecules into a broad spectrum of microorganisms.

14 8 Concise Monograph Series FIGURE 6. Agrobacterium naturally infects some types of plants and introduces novel DNA into them. Biologists use specially tailored forms of Agrobacterium to modify plants genetically. Isolate desired gene from donor organism Remove unwanted genes from plasmid Restriction enzyme cuts DNA at specific places Insert selected gene into cut plasmid DNA ligase joins DNA fragments Restriction enzyme cuts DNA at specific places Float leaf discs on a suspension of Agrobacterium Plasmid Bacterial chromosome Put novel plasmid into Agrobacterium tumefaciens Bacteria enter cells at cut edge and introduce novel plasmid into plant tissue Cultivate leaf discs on nutrient medium Regenerate whole plants, which now contain the introduced gene Genetically modified animals. The DNA of animals can also be modified by genetic engineering. It is necessary to introduce genes at an early stage of development if they are to be present in all of the cells of a mature animal and be passed on to its offspring. DNA can be injected into newly fertilized egg cells t h rough a very fine glass pipette. Only a small proportion of such injected eggs take up the new genes. The injected eggs are transferred into the uterus of a suitable foster mother. This is the only method so far

15 Food Biotechnology 9 FIGURE 7. DNA probes can be used to detect particular genes or fragments of DNA that have been isolated from organisms. This allows the presence of genetic characteristics to be confirmed rapidly, without the need to, for example, grow a plant to maturity. DNA fragments placed in these wells cdna or RNA probe added Probe revealed e.g. by chemiluminescence or autoradiography DNA is isolated from the test organism, and amplified if necessary The DNA is cut into fragments by a restriction enzyme DNA fragments migrate towards the positive electrode The fragments are separated by size in a slab of agarose gel The DNA is transferred to a nylon membrane The probe binds to complementary sequences of DNA The probe reveals the presence of DNA to which it has bound that works for cows, pigs, sheep and goats. Microinjection can also be used to introduce new genes into fish eggs, but it is not suitable for the eggs of birds. However, specially modified viruses have been used to introduce, for example, disease resistance into chickens. The viruses, which are made harmless, are inserted through the shell of the egg. Marker genes and gene probes. Whatever method is used, at best only a small proportion of treated cells take up the introduced DNA. Screening is therefore necessary to discover which cells have done so. As mentioned above, plasmids often carry genes which help the microbes that possess them break down particular antibiotics. These genes can be used as "markers" to identify those cells which have taken up plasmids, for when the cells are placed in a growth medium which contains an appropriate antibiotic, only those with plasmids will thrive. Several different types of marker genes exist within the plant kingdom, and these are being developed as alternatives to antibiotic markers. Other methods for identifying transferred genes include the PCR. This method enables the amplification of transplanted DNA sequences, which can then be detected using "gene probes". Gene probes are small fragments of singlestranded DNA or RNA which bind to complementary sequences in the DNA that is being sought out (Figure 7). Probes can also be used to detect the DNAof microorganisms that might contaminate food. They are so sensitive and specific that it is possible to use probes to differentiate between strains of the same species and to determine whether particular microorganisms are capable of producing toxins. Genetic switches. To ensure that the recipient cell's biochemical machinery will allow introduced genes to be expressed, sequences of DNA called control regions are required. One well-studied control region switches

16 10 Concise Monograph Series on and off a gene for making β-galactosidase. This enzyme enables bacteria to metabolise lactose, but it is p roduced only when that sugar is present in the surrounding medium. The "genetic switch" associated with the gene encoding β-galactosidase can be placed in front of other genes, so that the addition of lactose to broth in which the engineered cells are growing triggers expression of the new genes. There are several other genetic switches, some of which can even be thrown by a simple change in temperature. Other biotechnological methods Cell culture Many types of plant cells from a variety of species can be cultivated in vitro by supplying them with nutrients and growth substances under strict aseptic conditions (Figure 8). Illumination may also be required. Cultures of individual cells, grown in a fermenter, can be used in preference to whole plants for producing high-value products such as natural food colourings (for example, betanin, the red colour from beetroot) and flavourings (for example, vanilla and mint oil). Problems with this technique arise from the fact that most of these substances are produced only by mature cells that are not dividing. In culture, the cells tend to be actively growing, and so do not yield large quantities of these desirable products. A recent development with considerable promise is the cultivation of "hairy root cultures". By infecting plant tissues with Agrobacterium rhizogenes (a relative of A. tumefaciens), the production of root-like structures can be triggered. These transformed cells can be grown indefinitely on simple solid or liquid media. It is possible that they could be used to produce natural food flavourings and colourings without having to rely on plants grown in the open, which are subject to variations in availability and quality. However, work on hairy root cultures is still at the experimental stage. From undifferentiated cells (or callus) grown in vitro, whole plants can be regenerated by adjusting the p roportions of various growth factors in the surrounding medium. This enables the propagation of many thousands of plants from just a few cells, greatly accelerating the process of plant breeding. Although plants produced by cell culture should be genetically identical, the chromosomes in callus cells frequently undergo considerable rearrangement. Potato cells are especially prone to this. Such variation is a rich source of new strains, often with desirable characteristics. However, this phenomenon can be a serious drawback when great effort has been devoted to cloning a precious new plant variety. Problems of this nature beset a major project to grow oil palms in the mid-1980s. Cell culture is also used for the conservation of those plant varieties which cannot be maintained in a normal seed bank. Genetic changes are in this case highly undesirable. For some species, plantlets raised from cloned cells can be planted directly in farmers' fields. A l t h o u g h expensive, this method is especially suited to crops such as bananas, which do not have seeds. More often, plant cell culture is used to generate uniform, viru s - f re e plants of high value (such as ornamental species) or the plants from which seeds are subsequently obtained. Cauliflower seeds, for example, are often obtained from such stock. Cell culture can also be used to make "artificial seeds" cultured plant embryos that are subsequently coated by a protective layer of hard gel. The cost of producing such artificial seeds currently restricts their use to high value crops. However, the process is highly productive; one estimate suggests that from a 10-litre fermenter sufficient embryos could be produced to satisfy French farmers' entire annual demand for carrot seeds.

17 Food Biotechnology 11 FIGURE 8. Plants can be cultured on sterile solid or liquid media that contain nutrients and plant growth substances ("plant hormones"). Using this method, many thousands of identical plants can be produced from a small amount of plant tissue. Explants Buds Seeds Roots Nodal segments Preparation Surface sterilization Incubation Undifferentiated callus tissue Subculture Callus tissue Consecutives rinses in sterile distilled water Nutrient media containing plant growth substances Agitation LIGHT Cells in liquid suspension culture Nutrient medium The walls of plant cells are composed mostly of cellulose. A cellulase enzyme preparation can be used to remove these walls, leaving spherical pro t o p l a s t s bounded by a thin membrane. The absence of a cell wall makes protoplasts especially amenable to genetic engineering by electroporation or ballistic impregnation (see page 7). Animal cells may also be cultured in vitro, although it is not possible to grow whole animals or anything other than sheets of tissue from them. Their main use is in medical research, where, for instance, they are used to maintain viruses which cannot be cultured other than by infecting living cells. Cell fusion Plant cell protoplasts from different species can be fused to create complex "hybrids" with two or more nuclei. Even protoplasts derived from different genera can be joined; it does not matter that these plants are unable to interbreed. In theory, hybrid plants with characteristics from both donor cells can be regenerated from fused p rotoplasts. This could provide a simple route for introducing, say, the ability to fix atmospheric nitrogen from legumes into cereals. However, enthusiasm for this technique has waned since the first successful experiments during the 1970s. Protoplasts of many species, fused or not, have proved difficult or impossible to culture, let alone regenerate into entire plants. Cell fusion of a different type, using animal cells, has been more valuable. Antibodies can be produced by hybridomas grown in vitro. A hybridoma is an antibody producing cell (which is normally difficult to culture in isolation) fused with a tumour cell (which is virtually immortal in culture). Large quantities of specific antibodies (called monoclonal antibodies because they a re all identical) can be produced by cultivating hybridomas in a fermenter. These antibodies can be

18 12 Concise Monograph Series used in diagnostic tests of great sensitivity. For example, aflatoxins are potentially lethal compounds that are produced by fungi which contaminate stored food. Antibodies raised against aflatoxins are now used routinely in inexpensive and simple-to-use diagnostic kits. The same antibody technology is also used to detect the hormones associated with ovulation and p re g n a n c y. Such knowledge brings significant advantages to animal breeding programmes. FOOD PRODUCTION Microbial production of food, food additives and processing aids Fermented foods Fermented foods are foods in which desirable changes have been produced by the action of microbes or enzymes. Over different traditional fermented foods have been catalogued. They include the bread, yoghurt and cheese that are familiar in Europe and North America. In Africa, foods made from fermented starch crops (yams, cassava, etc.) are more important, whereas in Asia products derived from fermented soya beans or fish predominate. Fermented beverages include not only the obvious alcoholic drinks, but also tea, coffee and cocoa (fermentation of the leaves or beans occurs after tea, coffee or cocoa have been harvested). Fermentation can make the food more nutritious, tastier or easier to digest or can enhance food safety. Fermentation also helps preserve food and increase its shelf life, reducing the need for additives, refrigeration or other energy-intensive preservation methods. For several thousand years, traditional fermentation (such as brewing) has given people the opportunity to cultivate microbes on a large scale and in a safe manner. Single-cell protein. In the 1960s, protein from microbial sources (single-cell protein or SCP) was thought to have considerable potential, particularly for Third Wo r l d countries. Few of the early projects aimed to produce food for humans, aiming instead to provide nutritious, low cost animal feed. Unfortunately, most of the SCPorganisms (yeasts, fungi, and bacteria) used petrochemical derivatives as a source

19 Food Biotechnology 13 of carbon. Even when oil prices were low, the processes were only marginally economic. Consequently the oil price rises of the 1970s ended most SCP projects. One company had an efficient large-scale process using the bacterium Methylophilus methylotro p h u s that could utilize methanol to produce a partially purified protein for animal feed. However, the cost of soya or fishmeal for animal feed remained considerably lower than that of the bacterial protein. Consequently, production of the protein ceased in the late 1980s. Of all the SCP projects started in the 1960s, only one has survived to become a commercial success. Fungal protein (in the form of mushrooms and yeasts) has been accepted as food for generations. One company pioneered the production of fungal protein from the mycelium of Fusarium graminearum, which is cultivated on glucose made from maize starch. The food is marketed in the United Kingdom, Ireland, Germany, Switzerland and Belgium. By 1995, annual production is expected to reach tonnes enough for about 28 million meals for a family of four. Unlike soya products, the fungal protein has an excellent texture, attributed to a special linear arrangement of the fungal hyphae which is similar to that of muscle fibres in meat. It has a high protein and fibre content, yet contains almost no fat, which accounts for its popularity with health-conscious consumers. The product has little taste of its own, yet absorbs other flavours readily and can be combined with a wide range of ingredients. As a form of SCP, algae are of interest in subtropical and tropical regions, where sunlight can be utilized as an energy source. Often the production of algal protein is associated with fish farming. Algae of the genera Chlorella and Scenedesmus have been used as food in Japan, and Spirulina is being produced commercially in several countries, including the United States, Mexico and Israel. Often the product is sold as a high-value "health food". I m p roved microbial culture s. All SCP p rocesses use naturally occurring micro o rganisms that have been carefully selected from wild populations. Production of the fungal protein, for example, uses a strain of Fusarium g r a m i n e a r u m that was isolated from a soil sample obtained close to a research laboratory. Before that, many thousands of samples from around the globe had been laboriously screened to see whether they contained a fungus with a suitable nutritional profile, pattern of growth and other desirable properties. For the microbial production of a substance such as an amino acid, it is occasionally possible to find a rare natural variant or "mutant" which lacks a critical step in one of its biochemical pathways and consequently overproduces the desired material. Where such mutants are hard to find, they can sometimes be induced artificially, but such techniques are slow and rely heavily on chance. The goals of genetic modification applied to strain i m p rovement are essentially the same as those of traditional methods, but the techniques are more precise and far quicker. In addition, genetic modification allows the genetic "solutions" developed by nature to be transferred from one species to another, so that this species can also benefit from certain genetically determined traits, for example, disease resistance. Genetically modified yeasts. In 1990 the United Kingdom became the first country to permit the use of a live, genetically modified organism in food. This was a special strain of baker's yeast engineered to make bread dough rise faster. Existing genes were placed under the c o n t rol of stro n g e r, constitutive promoters, which helped the yeast break down sugar maltose faster than usual. Ordinary brewer's yeast (Saccharomyces cerevisiae) is able to utilize a variety of carbohydrates as an energy source. These include glucose, sucrose and maltose.

20 14 Concise Monograph Series Although sucrose is readily available (as cane or beet sugar), glucose and the other sugars must be prepared by the enzymic breakdown of starch (see Sweetener p ro d u c t i o n, below). Unlike S. cere v i s i a e, the closely related yeast S. diastaticus is able to grow on starch and dextrins because it makes an extracellular enzyme, glucoamylase, which catalyses the breakdown of starch. Saccharomyces diastaticus cannot be used directly for brewing because it produces a compound which gives beer a spicy flavour. Great interest has therefore focused on transferring the gene for glucoamylase from S. diastaticus into S. cerevisiae. Such a yeast would be better able to utilize the carbohydrate present in conventional feedstocks, which would increase the yield of alcohol and enable the production of a full-strength, lowcarbohydrate beer without the use of extra enzymes after the beer had been brewed. A modified yeast of this sort, produced by a re s e a rch foundation, re c e n t l y received approval for use in beer production in the United Kingdom. Food yeasts which have been genetically modified to metabolise a wider range of sugars also help reduce the levels of polluting waste in effluent. Sugar beet molasses is widely used as the main raw material in the production of baker's yeast. Beet molasses contains, in addition to sucrose, a small proportion of raffinose. This sugar is not fully broken down and utilized by the yeast, and the unused part (melibiose) is found in waste water from factories. The new strains of yeast utilize raffinose completely, enhancing the yield of baker's yeast and leading to a cleaner effluent. Improved starter cultures for the dairy industry. When cheese, yoghurt and similar dairy products are made it is important that only the desired microorganisms be allowed to ferment the milk. Failure to exclude unwanted organisms can lead to poor flavours, low yields and even food poisoning. Scrupulous hygiene is required, and often the milk is heat treated to kill all or some of its microbial flora. Starter cultures of only the desired microbes are then added to the milk in sufficient volumes and in the appropriate conditions to ensure their rapid growth. When improved starter cultures are developed, it is sometimes possible to choose between long-established, conventional techniques and the modern methods of genetic modification. However, the organisms that are produced may be identical, irrespective of the method chosen. One example where different routes led to the same result is a new yoghurt that keeps its fresh "home made" taste for several weeks without the risk of turning acidic and bitter. Normally, the starter culture bacteria in yoghurt turn the milk's sugar, lactose, into lactic acid within days. All that is needed to produce a yoghurt that stays mild is a Lactobacillus strain that cannot metabolise lactose. Such variants already exist in nature. One possibility is t h e re f o re to search for these organisms in nature. Another conventional approach consists of treating the lactobacilli in various ways to increase their mutation rate in the hope that the desired trait will accidentally be created. A third option involves modern genetic techniques: identifying the precise gene (or genes) responsible for lactic acid production, isolating and modifying them to inactivate lactic acid production, and then replacing them in the bacteria. All three techniques have proved successful and led to identical mild-tasting yoghurts. In many other cases, however, only the "designer gene" approach can deliver the desired results. Although this work is still at the laboratory stage, starter cultures for yoghurt and cheese production have now been altered to provide built-in p rotection against other microbes that cause food poisoning. The starter cultures have been modified so

21 Food Biotechnology 15 that they produce a compound which breaks down the cell walls of the potentially lethal food poisoning o rganism Listeria monocytogenes. Modified bacteria could also be put into other foods as a fail-safe mechanism in case the food is stored in the wrong conditions. It might also be possible to design foods that could be protected against a range of other foodpoisoning organisms, such as Salmonella. Other modified dairy starter cultures under development produce flavour compounds for bettertasting products and are able to resist attack from viruses that would otherwise ruin the production of yoghurt, cheese and similar products. Food additives and processing aids For many years, a wide range of food additives, supplements and processing aids have been obtained from microbial sources. These include amino acids, citric acid, vitamins, natural colourings and gums as well as enzymes. The production microorganisms have been selected from nature to ensure that, for example, they produce high yields of a good-quality product and/or are easy to grow or that the product is easy to separate and purify. Laborious screening and selection p rocesses are now being augmented by modern genetics, allowing quicker, more precise selection and improvement of existing production strains. Amino acids. In foods, amino acids are used to enhance flavours and to act as seasonings, nutritional additives and improvers (in flour). They are used in both human food and animal feed. Bacteria or fungi which have been specially selected to overproduce specific amino acids are grown in large fermenters. The acids are secreted into the fermentation medium and harvested. The most important commercial products are glutamic acid, which is used as monosodium glutamate, a flavour enhancer; lysine, cysteine and methionine, which are used as supplements in animal feeds that are usually deficient in these essential amino acids; and phenylalanine, which is used in animal feed and in the m a n u f a c t u re of the sweetener aspartame (see Sweeteners, below). Most of the 20 amino acids needed to make proteins are produced by fermentation in hundred- or thousandtonne quantities. In 1990, a company made a batch of tryptophane that was subsequently implicated in a rare degenerative disord e r. Concern arose because the tryptophane had been produced by a genetically engineered strain of Bacillus. However, it is thought that the illness was caused by insufficient purification of the tryptophane and not by the genetic modification itself. Gums. Several gums produced by microorganisms and plants are used widely in the food industry as thickeners, emulsifiers and fillers. Recently a process was developed to turn relatively cheap guar gum (obtained from seeds) into something akin to the more expensive locust bean gum. An enzyme (αgalactosidase) from the guar seeds is responsible for this transformation. The gene encoding the enzyme was inserted into baker's yeast, and α-galactosidase can now be produced in quantity. Production and processing of α-galactosidase by modified organisms brings several other benefits (Table 1). Bacterial polysaccharides currently occupy only a small fraction of the food ingredients market, but genetically modified cells could produce a wide range of novel gums with improved properties. Sweeteners. Aspartame, a peptide which is 160 times sweeter than sucrose, is now used in an increasing range of foods and beverages. Aspartame's sweetness was (allegedly) discovered by accident when James Schlatter, working in the United States, licked his fingers to separate a stack of papers. He had previously spilt some aspartame on his hands while working in the

22 16 Concise Monograph Series TABLE 1 A comparison of production and downstream processing requirements of α-galactosidase produced by standard and modified yeasts Standard yeast Modified yeast Production Yeast required 236 tonnes 10 tonnes Waste water tonnes 90 tonnes Waste yeast 400 tonnes 12 tonnes Downstream processing Ammonium sulphate tonnes 25 tonnes Potassium sulphate 25 tonnes 1 tonne Aluminium gel 1 tonne None Filtration aid 133 tonnes 5 tonnes Solid waste 540 tonnes 18 tonnes Liquid waste tonnes 26 tonnes Demineralized water m 3 50 m 3 Iced water m m 3 Energy kw kw Steam 220 tonnes 50 tonnes laboratory. Chemists at another laboratory in the United Kingdom had independently synthesized aspartame some years before but had failed to notice its sweetness. The original manufacturing process linked the two amino acids phenylalanine and aspartic acid (both products of fermentation) by chemical means. Today a more efficient enzymatic method has been developed. Other food additives from microbes. Citric, acetic, lactic and ascorbic acids are produced in large volumes for the food industry by microbial fermentation. Enzymes. A wide range of microbial enzymes are used by the food industry. Some of the more important applications are shown in Table 2. Plant biotechnology Plant breeding techniques Since the origins of agriculture some years ago, farmers have been trying to improve their cro p s. Initially, people simply replanted some of the seeds f rom their best plants each year. Crosses between chosen individuals of the same species led to gradual improvements in the stock. Ancient South American civilizations relied on sweet corn (maize), the product of a cross between two dissimilar wild plants, for much of their diet. For thousands of years this species has been totally dependent on human intervention because it has no natural mechanism for dispersing its closely bound seeds. Around the turn of the century the scientific

23 Food Biotechnology 17 TABLE 2 Uses of enzymes in the food industry Enzyme Product Use α-amylase Sweeteners Liquefaction of starch Beer Removal of starch haze Bread, cakes and biscuits Flour supplementation Amyloglucosidase Sweeteners Saccharification Low-carbohydrate beer Saccharification Wine and fruit juice Starch removal Bread manufacture Improved crust colour β-galactosidase (lactase) Whey syrup Greater sweetness Lactose-reduced milk and dairy Removal of lactose for those who products are lactose intolerant Ice cream Prevention of "sandy" texture caused by lactose crystals Chymosin (rennin) Cheese Coagulation of milk proteins Glucose isomerase High-fructose syrup Conversion of glucose to fructose Glucose oxidase Fruit juices Removal of oxygen Invertase Soft-centred sweets Liquefaction of sucrose Sugar syrups Lipases Cheese Flavour development Accelerated ripening Flavourings Ester synthesis Papain Beer Removal of protein Pectinases Wine and fruit juice Increased yield, clarification Coffee Extraction of the bean Proteases (various) Dairy products Modification of milk proteins Caviar Viscosity reduction of "stickwater" Bread, cakes and biscuits Gluten weakening Meat Tenderisation Removal of meat from bones